2'-deoxycytidines carrying amino and thiol functionality: synthesis and incorporation by Vent (exo-) polymerase.

Article abstract of DOI:10.1021/ol0360290

[reaction: see text] The synthesis of 2'-deoxycytidine nucleosides bearing amino and thiol groups appended to the 5-position of the nucleobase via a butynyl linker is described. The corresponding triphosphates were then synthesized from the nucleoside and incorporated into oligonucleotides by Vent (exo(-)) DNA polymerase. The ability of Vent (exo(-)) polymerase to amplify oligonucleotides containing these functionalized cytidine derivatives in a polymerase chain reaction (PCR) was demonstrated for the amino-functionalized derivative.

Full text of DOI:10.1021/ol0360290

ORGANIC
LETTERS
2004
Vol. 6, No. 4
489-492
2′-Deoxycytidines Carrying Amino and
Thiol Functionality: Synthesis and
Incorporation by Vent (Exo-)
Polymerase
Abhijit Roychowdhury,† Heshan Illangkoon,† Cynthia L. Hendrickson,‡ and
,†,‡
Steven A. Benner*
Department of Chemistry, UniVersity of Florida, P.O. Box 117200,
GainesVille, Florida 32611, and Department of Anatomy and Cell Biology,
UniVersity of Florida, P.O. Box 100215, GainesVille, Florida 32610
benner@chem.ufl.edu
Received October 16, 2003
ABSTRACT
The synthesis of 2′-deoxycytidine nucleosides bearing amino and thiol groups appended to the 5-position of the nucleobase via a butynyl
linker is described. The corresponding triphosphates were then synthesized from the nucleoside and incorporated into oligonucleotides by
Vent (exo-) DNA polymerase. The ability of Vent (exo-) polymerase to amplify oligonucleotides containing these functionalized cytidine derivatives
in a polymerase chain reaction (PCR) was demonstrated for the amino-functionalized derivative.
Deoxyribonucleosides carrying functionality appended at the
5-position of uracil were introduced three decades ago to
complement the functionality that DNA carries intrinsically.1
Fluorescent appendages attached to 2′,3′-dideoxynucleotides
have been the key to automated DNA sequencing.2 Func-
tionalized appendages may also increase the power and
versatility of nucleic acids as receptors, ligands, and cata-
lysts.3 Modified standard nucleotides have been incorporated
into in Vitro evolution experiments.4
The C5-position of the pyrimidine nucleobases is an
appropriate place to introduce functionality, as the site lies
in the major groove of the duplex, where appendages do not
interfere with Watson-Crick pairing. Appendages at this site
are also well tolerated by RNA and DNA polymerases, which
do not interact with the nucleobases in the major groove.4c,5,6
Most of the previous work in this area has functionalized
2′-deoxyuridine, although some work has also appended
functionality through the 5-position of 2′-deoxycytidine
analogues.7 We report here the synthesis of 2′-deoxycytidine
nucleosides carrying amino or thiol terminal functionality
appended at position C5 via a four-carbon alkynyl linker.
We also report the successful incorporation of these as
triphosphates into DNA using Vent (exo-) DNA polymerase
and PCR amplification.
† Department of Chemistry.
‡ Department of Anatomy and Cell Biology.
(1) (a) Bergstrom, D. E.; Ruth, J. L. J. Am. Chem. Soc. 1976, 98, 1587-
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Sci. U.S.A.: Biol. Sci. 1981, 78, 6633-6637.
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Recent reports suggest that protected 2′-deoxycytidine
nucleosides carrying a C-5 acetylene group readily suffer
10.1021/ol0360290 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/17/2004

unwanted cyclization.4b Thus, we explored chemistry to
attach substituents to the 5-position of an unprotected cytidine
nucleoside.
Scheme 2a
But-1-yne-4-thiol (2) having its thiol group protected as
the disulfide with tert-butylthiol was synthesized from but-
3-yn-1-ol 1 as reported previously.8 The corresponding
1-aminobut-3-yne, with the amino group protected as the
trifluoroacetyl amide, was synthesized from but-3-yn-1-ol
via a route adapted from the literature.9 Here, the free
hydroxyl group of 1 was converted to the corresponding
azide via mesylation. The azide was then reduced to the
corresponding amine with PPh3. The reaction mixture was
acidified, the side products were extracted, and the free amine
(5) was isolated by the neutralization of its acid salt. Reaction
with trifluoroacetic anhydride gave 6 in 51% yield (Scheme
1).
a Conditions: (a) DMTCl/TEA/DMAP/Py anhydrous/6 h/0 °C
to rt; (b) 2 or 6/Cul/TEA/Pd(PPh3)4/DMF anhydrous/10-14 h/rt;
(c) Di-t-butyl-1-(t-butylthio)-1,2-hydrazine-dicarboxylate/LiOH/
MeOH-THF/90 min anhydrous. (d) Py/DMAP/TEA/Ac2O/4 h/0
°C to rt.
Scheme 1a
The benzoyl-protecting group on 9 was replaced by the
more stable tert-butylthio group8 in a one-step reaction with
di-t-butyl-1-(t-butylthio)-1,2-hydrazine-dicarboxylate in the
presence of LiOH to give 11. Acetylation of 10 and 11
yielded mixtures of both di- and monoacetylated products,
12 and 14, 13 and 15, respectively, with the major products
being the desired 14 and 15.
We attempted to selectively deacetylate 12 and 13 using
ZnBr2.11 An equimolar amount of the Lewis acid gave the
desired 14 and 15, together with the unreacted starting
compound 12 and 13, in approximately equal amounts.
Treatment with 2 equiv of ZnBr2 yielded the N-4 deacety-
lated and deprotected 5′-OH compounds 16 and 17 only.
Compounds 16 and 17 were also prepared from 14 and 15
using the commercial deprotecting mixture (2.5% trichlo-
roacetic acid in CH2Cl2) for oligonucleotide synthesis via
solid-phase chemistry (Scheme 3).
a Conditions: (a) PPh3/DIAD/THF/TEA/14-18 h/rt; (b) MsCl/
anhydrous ether/3 h/0 °C/H2O; (c) NaN3/anhydrous DMF/3.5 h/67
°C/H2O; (d) PPh3/anhydrous ether/H2O/18 h/0 °C to rt/10% HCl
(aq); 10% NaOH (aq); (e) anhydrous MeOH/CF3CO2CH3/18 h/0
°C to rt.
Sonogashira coupling10 of the compounds with the 5′-
tritylated 5-iodo-2′-deoxycytidine (8) in the presence of
palladium(0) catalyst gave the desired products (9 and 10)
(Scheme 2).
The 5′-tritylated nucleoside was used in preference to the
5′-hydroxy nucleoside because the former is easier to handle
(with respect to monitoring and its greater solubility) and
saves solvents during column chromatography purification.
Scheme 3a
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a Conditions: (a) ZnBr2/MeOH/CHCl3/2 h/rt; (b) TCA in CH2Cl2/
30 min/rt.
Triphosphate synthesis was carried out following literature
procedures.12 Thus, reaction of 16 and 17 with POCl3 in
490
Org. Lett., Vol. 6, No. 4, 2004

trimethyl phosphate and proton sponge gave the phospho-
rodichloridates as intermediates, which were converted in
situ with pyrophosphate to the corresponding triphosphates.
Hydrolysis with NH4OH gave the desired nucleotide
triphosphates, 18 and 19 (Scheme 4).
Scheme 4a
Figure 1. Primer extension experiment using amino-functionalized
2′-deoxycytidine triphosphate 19. Denaturing 20% PAGE-urea gel
was used. 5′-Radiolabeled primer was visualized by a phospho-
imager. All lanes contain annealed primer and template, buffer,
and water. Lane 1: All natural dNTPs (0.1 mM each), no
polymerase (negative control). Lane 2: Vent (exo-) DNA poly-
merase and dCTP only showing pausing after incorporation of dC.
Lane 3: Vent (exo-) polymerase and natural dNTPs showing
formation of full-length product (positive control). Lane 4: Vent
(exo-) polymerase and 19 only, showing incorporation of a single
19; lower mobility due to positively charged functionality. Lane 5:
Vent (exo-) polymerase and 19, dTTP, dGTP, dATP, expecting
incorporation of three 19s.
a Conditions: (a) POCl3/(MeO)3P(O)/proton sponge/2.5 h/0 °C;
(b) Tri-n-butylammonium pyrophosphate/n-tributylamine/DMF/
TEAB/2 min/0 °C to rt; (c) NH3/rt/18 h.
The ability of the functionalized dCTP analogues (18 and
19) to serve as substrates for thermostable DNA polymerases
under PCR conditions was then studied. Since different
polymerases often behave differently with unnatural nucleo-
sides,4h representatives of two evolutionary families of DNA
polymerases13 were examined. These were the Taq DNA
polymerase from Thermus aquaticus (representing Family
A) and Vent (exo-) DNA polymerase from Thermococcus
litoralis (representing Family B).
Primer extension experiments were done using PAGE-
purified 5′-[γ-32P]-radiolabeled primer (5′-GCG TAA TAC
GAC TCA CTA TAG-3′) and template (5′-GAC ACG CGC
TAT AGT GAG TCG TAT TAC GC-3′) (both from
Integrated DNA Technologies, Coralville, IA).
Taq incorporated neither 18 nor 19 with acceptable
efficiency (data not shown). Vent (exo-) DNA polymerase
incorporated both 18 and 19. Thus, Vent polymerase
incorporated 19 (Figure 1; Lane 6) opposite G in a single
base extension. Full-length product was obtained upon
addition of a complete set of natural triphosphates excluding
dCTP (Figure 1; Lane 5).
Similarly successful incorporation was observed for ana-
logue 18 with protected thiol functionality (data in Supporting
Information).
Compound 19 carrying a free amino group successfully
replaced dCTP in PCR amplification using Vent (exo-)
polymerase (Figure 2; Lane 6). This PCR required incorpo-
ration of 22 and 31 functionalized cytidine analogues per
strand in the forward and reverse reactions.
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Figure 2. PCR amplification with 19 replacing dCTP. Agarose
gel (2%) was used and stained with ethidium bromide. A total of
25 PCR cycles were run, with 2 min each incubation. Lane 1:
Promega DNA ladder 25-300 nts. Lane 2: Negative control,
lacking polymerase. Lane 3: Positive control with standard dNTPs.
Lane 4: Positive control including both 2′-dCTP and 19 (1:1 ratio)
and standard dNTPs. Lane 5: With 2 µM 19 and standard dNTPs.
Lane 6: With 4 µM 19 and standard dNTPs. Template (100mer):
5′-CGC ATT ATG CTG AGT GAT ATC TAT CCA GAC CTA
GAA AGA GTG CAC TGA TGC TGT TCG AGC GCA CGG
CCT CCA ACA TGC CGT CCA TGC ACC ACT AGA CCT C-3′.
Primer (24mer): 5′-GAG GTC TAG TGG TGC ATG GAC GGC-
3′. Reverse primer (24mer): 5′-CGC ATT ATG CTG AGT GAT
ATC TAT-3′.
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Org. Lett., Vol. 6, No. 4, 2004
491

Relatively low concentrations of triphosphate (2 µM) were
used in the PCR amplification. Compound 19 alone sup-
ported the synthesis of full-length products at these concen-
trations (Figure 2; lane 5), although doubling the concen-
tration of 19 increased the amount of full-length product
formed. This suggested that 19 was able to support PCR
amplification, but at modestly lower efficiency. Consistent
with this conclusion, a 1:1 mixture of 19 and dCTP in a 1:1
mixture showed reduction in the amount of full-length
product observed relative to dCTP alone. As a caveat, it is
important to note that these results are consistent with, but
do not absolutely prove, the presence of the functionalized
cytidine derivative in the product.
Two features of these results are particularly noteworthy.
First, in primer extension experiments, DNA carrying 3 equiv
of 19 showed a double band.
Polymerases frequently add a nontemplated nucleotide to
the product, and this is, we believe the simplest explanation
for this observation.14
rejected this hypothesis. Rather, it seems that the replacement
of dCTP by 19 simply lowers the yield of full-length product.
The accessibility of these functionalized nucleoside deriva-
tives and polymerases that accept their triphosphates suf-
ficiently well to support the polymerase chain reaction gives
two new tools to those wishing to perform molecular biology
with DNA containing thiol and amino functionalities. This
is another step toward the development of a synthetic
biology.15
Acknowledgment. We are grateful to Dr. Jodie V.
Johnson and the personnel at the mass spectroscopy lab at
the Department of Chemistry for acquisition of the mass
spectra. Useful discussions with Drs. C. R. Geyer, T. R.
Battersby, S. C. Jurczyk, and H. A. Held are acknowledged.
Extensive help in the HPLC purification by Dr. A. P. Kamath
is highly acknowledged. This work was partially supported
by grants from the Department of Defense 6402-202-LO-B
and the National Science Foundation (CHE-0213575).
Further, although 19 supported the PCR amplification of
DNA, the yield appeared to be lower than with standard
dCTP. We considered the possibility that the positively
charged amine groups may disrupt the intercalation by the
similarly cationic ethidium bromide. As the intensity of the
band increases when the concentration of 19 is doubled, we
Supporting Information Available: Experimental pro-
cedures and characterization of all new compounds along
with NMR spectral data. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL0360290
(14) Hu, G. DNA Cell Biol. 1993, 12, 763.
(15) Benner, S. A. Nature 2003, 421, 118.
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